HALTERES, Volume 4, 68-78, 2013

ISSN 0973-1555 © HIMENDER BHARTI, NAVPREET DHIMAN, MEENAKSHI BHARTI AND AIJAZ AHMAD WACHKOO

68

SEM studies on immature stages of Aphaenogaster beesoni

Donisthorpe, 1933 (Hymenoptera: Formicidae)

Himender Bharti1, Navpreet Dhiman, Meenakshi Bharti

2 and Aijaz Ahmad Wachkoo

3

Department of Zoology & Environmental Sciences, Punjabi University, Patiala - 147002, India

(e-mail: 1himenderbharti@gmail.com;

2adubharti@yahoo.co.in;

3aijaz_shoorida@yahoo.co.in)

Abstract

Larval instars of Aphaenogaster beesoni Donisthorpe, 1933 are herein described in detail by scanning electron

microscopy. Based on the body profile, body size, types of body hairs and head capsule size a total of five

larval stages are recognised. All five instars show clear variations in their body shape and mouth parts. Body

of the first instar is Aphaenogastroid, while that of the second, third and fourth is Pogonomyrmecoid, and in

fifth it again becomes Aphaenogastroid type.

Keywords: SEM, immature stages, Aphaenogaster, India.

Introduction

The ant genus Aphaenogaster Mayr

includes 180 extant species, 45 subspecies and

19 fossil species; these ants are widely

distributed in all geographical regions of the

world except Ethiopian region (Bolton et al.,

2007; Bolton, 2013). The genus includes one of

the most primitive and generalized ant species

with fossil records in the Baltic amber.

Currently, it is represented by 176 extant species

from the World of which 14 belong to Indian

region (Bharti, 2011).

The significance of larval descriptions to

ant systematics has been discussed in a number

of sources (Wheeler and Wheeler 1976, 1986,

1988; Fox et al., 2007, 2010, 2012; Solis et al.,

2007, 2010a, b, 2011; Jesus et al., 2010;

Nondillo et al., 2010; Bharti and Gill, 2011 and

Bharti and Kaur, 2011). As rightly put by

Wheeler and Wheeler (1976), “Modern

taxonomy is complete only when variety of data

is pushed as far as possible towards the limit of

practicability. The object of classification should

be holomorph i.e. studying all the characteristics

of an individual throughout its life”. Apart from

their importance in the study of general biology

(Peeters and Holldobler, 1992), the larval

characters of ants have also been used for

phylogenetic and behavioural studies (Petralia

and Vinson, 1979a, b; Schultz and Meier, 1995;

Masuko 2003, 2008; Pitts et al., 2005 and

Signorotti et al., 2013).

Aphaenogaster beesoni Donisthorpe,

1933, a high altitude species is widely

distributed in Northwest Himalaya. It nests

under stones and is found mainly in forested

areas with scarce undergrowth and fairly moist

surfaces. Intensive ecological and taxonomic

studies have been made on Aphaenogaster

beesoni but there is a void in our knowledge, as

far as its larval morphology is concerned.

Here we provide reliable distinguishing

larval characters, to fill this gap in our

understanding of Aphaenogaster immatures. The

present study marks the first ever comprehensive

study on immatures of Aphaenogaster beesoni.

Based on ultrastructural images, minute

Himender Bharti, Navpreet Dhiman, Meenakshi Bharti and Aijaz Ahmad Wachkoo

69

morphological details of different larval forms

have been studied, to generate baseline data and

to aid in future interspecific diagnosis.

Materials and Methods

To study the immature stages of

Aphaenogaster beesoni, larval forms were

collected from 5 different colonies located at

Solang valley (2560m), Himachal Pradesh,

India. The larvae were fixed in the Dietrich’s

solution for 24-48 hrs, and then preserved in

80% alcohol. The larvae were separated into five

instars on the basis of body profile, body size,

types of body hairs and head capsule size.

After separation of larval forms, all instars

(n=10) were prepared for scanning electron

microscopy using following steps: a) instars

preserved in 80% alcohol were post fixed in 1%

Osmium tetraoxide and then placed in

refrigerator for 2 hours; b) after post fixing, the

specimens were dehydrated in a graded acetone

series; c) specimens were critical point dried in

desiccators; d) dried specimens were then

attached to the aluminium stubs with double

faced conductive adhesive tape; e) specimens

were then placed in the sputter for coating with

the palladium and f) specimens were studied

under a Zeiss EVOMA10 Scanning Electron

Microscope at 20 KV/EHT.

The terminology given by Wheeler and

Wheeler (1976) and Fox et al. (2007) has been

used to describe the larvae of Aphaenogaster

beesoni. As body profile of Aphaenogaster

beesoni is of curved type, it is therefore

measured as cursor width and cursor height.

Body hairs are measured at full length. Head

capsule width, mouthparts and other

morphological characters are measured for at

least twenty individuals per larval instars.

Results

The detailed description of important

morphological larval characters of different

instars of Aphaenogaster beesoni is as follows:

First larval instar

Body: Whitish, Aphaenogastroid in shape

head bent ventrally, anus slightly subterminal

transverse slit with 10 body spiracles (Figs. 1a,

1b). Head and body covered with abundant

hairs. Mainly two types of hairs present on body

surface: smooth, unbranched flexuous hairs and

anchor-tipped hairs (Fig. 1c). Body length

865.3µm, width 564.1µm; body diameter at

thorax region 246.6µm, at abdomen region

336.4µm (Fig. 1a).

Head capsule: Cranium 224.9µm high and

278.8µm wide; roughly subcircular in shape

(Fig.1d). Head surface smooth with bilaterally

symmetrical tip-bifid hairs.

Mouthparts: Clypeus not clearly delimited

from the cranium, upper surface of clypeus

smooth, without sensilla, with two types of

hairs: simple and tip-bifid; labrum bilobed (Fig.

1e). Mandibles simple, sharp pointed;

Pogonomyrmecoid in shape, 92.38µm long and

32.55µm wide from the base with three medial

teeth lying approximately in same plane (Fig.

1f).

Second larval instar

Body: Body shape changed from

Aphaenogastroid in first instar to

Pogonomyrmecoid in second larval instar. In

this case the diameter is greatest near middle of

abdomen, decreasing gradually toward head and

more rapidly toward rounded posterior end;

thorax more slender than abdomen, forming a

ventrally curved neck (Fig. 2a). Body hairs are

less as compared with the first instar larva and

are of two types: deeply bifid measuring about

146.5µm and flexuous i.e. whiplike or

flagelliform, measuring about 154.6µm (Fig.

2b); anus subterminal in position (Figs. 2c, 2d).

Body length 1.403mm; width 949.7µm; body

diameter at thorax region 300.7µm, at abdomen

region 629.0µm (Fig. 2a).

Head capsule: Cranium 230.7µm high and

290.5µm wide; antennae distinct in diameter.

SEM studies on immature stages of Aphaenogaster beesoni Donisthorpe (Hymenoptera: Formicidae)

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Figures 1a-f: First larval instar; Aphaenogaster beesoni 1a. Body profile; 1b. Abdominal spiracles;

1c. Body hairs; 1d. Head cranium; 1e, 1f. Head and mouth parts.

Himender Bharti, Navpreet Dhiman, Meenakshi Bharti and Aijaz Ahmad Wachkoo

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Figures 2a-f: Second larval instar; Aphaenogaster beesoni 2a. Body profile; 2b. Body hairs;

2c. Anal region; 2d. Body hair and anal region; 2e. Cranium and thorax; 2f. Mouth parts.

SEM studies on immature stages of Aphaenogaster beesoni Donisthorpe (Hymenoptera: Formicidae)

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Head hairs which include simple, straight and

tip-bifid hairs are present on the occipital border,

on the vertex, on the genal region and on the

gula (Fig. 2e).

Mouthparts: Labrum bilobed; mandibles

Pogonomyrmecoid i.e. subtriangular in shape,

with three conspicuous medial teeth lying in the

same plane, mandibular surface spinulose.

Maxillae paraboloidal, long, wide with widely

spaced setaceous sensilla. Galeae simple

28.25µm long × 10.35µm wide. Labium stout

and hemispherical with some scattered setaceous

sensilla over the surface. Clypeus clearly

delimited from the cranium (Fig. 2f).

Third larval instar

Body: Body profile Pogonomyrmecoid

(Fig. 3a), anus subterminal in position. Body

hairs of three types: straight, deeply bifid and

dichotomously branching (Fig. 3b), more dense

on the posterior surface of body. Spiracles about

1.10µm in diameter, unadorned peritreme (Figs.

3c, 3d). Total body length 677.2 µm, from

thorax to anus 782.2 µm; body diameter at

thorax 444.9µm, at abdomen 625.6µm (Fig. 3a).

Head capsule: Cranium 250.6µm high and

298.5µm wide. Hairs mainly two type: simple,

slightly curved, long and tip-bifid. Antennae

distinct, shallow pits on upper half of cranium

with three sensilla (Fig. 3e).

Mouthparts: Clypeus not delimited from

the cranium, surface smooth, sensilla absent.

Labrum bilobed, with setaceous sensilla over the

anterior surface. Mandibles wide, subtriangular,

102.9µm long × 28.61µm (Fig. 3f).

Fourth larval instar

Body: Body profile Pogonomyrmecoid

(Fig. 4a), anus subterminal in position (Fig. 4b);

body hairs of three types: smooth flexuous; tip

bifid and deeply bifid; hairs less dense as

compared to the other instars. Body length 1.980

mm, width 932.6µm (Fig. 4a).

Head capsule: Cranium 244.0µm high ×

255.2µm wide (Figs. 4c, 4d). Hairs on the head

are of two types: simple, slightly curved and tip

bifid.

Mouthparts: Clypeus delimited from the

cranium, two rows of simple slightly curved and

tip bifid hairs present on the head; labrum

bilobed with setaceous sensilla over the anterior

surface. Mandibles long, subtriangular.

Maxillae paraboloidal in shape. Galeae 25.94

µm long × 12.05 µm wide (Fig. 4e).

Fifth larval instar

Body: Body Aphaenogastroid i.e. diameter

increasing gradually towards the middle of

thorax and abdomen; thorax arched ventrally but

not forming a distinct neck; posterior end

broadly rounded (Fig. 5a). Body hairs of two

types: simple 103.4µm long and slightly curved

at the tip 123.5µm long (Fig. 5b); anus

subterminal 415.4µm in diameter (Fig. 5c);

diameter at waist 654.2µm, at thoracic region

959.7µm and at abdominal region 1084.0µm

(Fig. 5a).

Head capsule: Cranium 763.2µm high ×

985.9µm wide from upper view and 221.0µm

high × 317.8µm wide from side view (Figs. 5d,

5e). Antennae present on the upper cranium

measuring 44.13µm long (Fig. 5e). Hairs

slightly curved and less in number (Fig. 5e).

Mouthparts: Clypeus delimited from the

cranium. Hairs almost absent from mouth region

(Fig. 5f).

Discussion

The present study marks first ever

comprehensive study on immatures of

Aphaenogaster beesoni. Using scanning electron

microscopy, the larvae of species were separated

into five instars; their minute details were

observed and later described in detail. This

division of larvae into 5 instars is carried for the

very first time (Table 1 and 2).

The body profile of 1st and 5

th larval instar

is Aphaenogasteroid and that of 2nd

, 3rd

and 4th

larval instar is Pogonomyrmecoid type (Figs. 1a,

Himender Bharti, Navpreet Dhiman, Meenakshi Bharti and Aijaz Ahmad Wachkoo

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Figures 3a-f: Third larval instar; Aphaenogaster beesoni 3a. Body profile; 3b. Body hairs; 3c. Body

spiracles; 3d. Spiracle with measurement; 3e. Cranium and sensilla; 3f. Mouth parts.

SEM studies on immature stages of Aphaenogaster beesoni Donisthorpe (Hymenoptera: Formicidae)

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Figures 4a-e. Fourth larval instar; Aphaenogaster beesoni 4a. Body profile; 4b. Anal region;

4c. Thorax and cranium; 4d. Head capsule; 4e. Galeae with measurement.

Himender Bharti, Navpreet Dhiman, Meenakshi Bharti and Aijaz Ahmad Wachkoo

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Figures 5a-f: Fifth larval instar; Aphaenogaster beesoni 5a. Body profile; 5b. Body hairs;

5c. Anal region; 5d. Head capsule; 5e. Antennae with measurement; 5f. Mouth region.

SEM studies on immature stages of Aphaenogaster beesoni Donisthorpe (Hymenoptera: Formicidae)

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2a, 3a, 4a, 5a respectively). The body size of all

larval instars increases as they grow. The

diameter from thorax and abdomen region in all

instars measured corresponds to 246.6µm :

336.4µm in 1st larval instar, 360.7µm : 629.0µm

in 2nd

larval instar, 444.9µm : 635.6µm in 3rd

larval instar, 775.6µm : 932.6µm in 4th larval

instar and 959.7µm : 1084.0µm in 5th larval

instar (Figs. 1a, 2a, 3a, 4a, 5a). Anus shifts from

slightly ventral in position in first instars to

subterminal in rest of the larval instars (Figs. 2c,

2d, 4b, 5c).

Hair type is one of the characters

considered in calculating the “specialization

indices” proposed by Wheeler and Wheeler

(1986). The presence of bifurcations in the head

hairs is being recently proposed as a character of

considerable importance for separating species

in the genus Solenopsis Westwood (Pitts, 2005).

Fox et al. (2007) pointed out that intraspecific

variation in types of head hairs might occur in

other ant species as well; the present study is in

accordance with the above study and suggests

the revision of all the previously described ant

larvae. Two types of hairs observed on the body

and head surface of immature larval instars

include:

Simple Hairs: Present on body and head

surfaces of almost all larval instars and are

further of two subtypes:

(i) Slightly curved or straight, present on head

and body of all instars.

(ii) Flexuous, present on the body of 1st and 2

nd

larvae and absent on head region.

Bifid hairs: These also have two subtypes:

(i) Tip bifid, observed on head surfaces of

almost all instars but were only present on the

body surfaces of 1st and 3

rd larval instars.

(ii) Deeply bifid, branched long and flexuous

hairs, observed on body surfaces of all instars

(Figs. 1c, 2e, 2f, 3c, 3e, 4d, 5b).

Size of cranium of larval instars increases

as they grow in size; measurements of cranium

of all instars corresponds to 224.9µm high ×

278.8µm wide in 1st larval instar, 234.6µm high

× 289.6µm wide in 2nd

larval instar, 239.0µm

high × 294.4µm wide in 3rd

larval instar,

244.0µm high × 305.2µm wide in 4th larval

instar and 251.0µm high × 317.8µm wide in 5th

larval instar (Fig. 1d, 2e, 3e, 4c, 5d).

Clypeus is not clearly delimited from the

cranium in all larval instars. Labrum is bilobed

in all larval instars. Mandibles are simple, sharp,

pointed and Pogonomyrmecoid in shape with

three medial teeth approximately in same plane

and increase in size from 1st larval instar to the

5th larval instar. Mandible measured in the first

larval instars is 92.38µm long × 32.55µm wide

from base. In third larval instar it is measured as

102.9 µm long × 38.91µm wide from base (Figs.

2f, 3f).

Maxillae and maxillary palps of all the

five instars are found to be very distinct. As the

growth proceeds in larvae the mouth parts also

increase in size. Galeae measurements of 2nd

and

4th larvae are 28.25µm long × 10.35µm wide and

about 35.94µm long × 12.05µm wide (Fig. 2f,

4e).

The details added to the larval description

of Aphaenogaster beesoni will provide an

insight into minute details of immature stages

and aid in future interspecific diagnosis.

Table 1: Hair types observed in larvae of Aphaenogaster beesoni.

Hair type Subtypes Description Location on instars (L)

Body Head Simple (S) S1 Slightly curved/straight All instars All instars

S1 Flexuous L1*, L2* Absent

Bifid (B) B1 Tip bifid L1*, L3* All instars

B2 Deeply bifid branches long and flexuous All instars Absent

* = (L1) First instar, (L2) Second instar and (L3) Third instar

Himender Bharti, Navpreet Dhiman, Meenakshi Bharti and Aijaz Ahmad Wachkoo

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Table 2: Body measures of different instars of Aphaenogaster beesoni.

Acknowledgements

The SEM lab facilities provided by Dr.

V.V. Ramamurthy (Principal Scientist), Division

of Entomology, IARI, New Delhi are gratefully

acknowledged. We are also thankful to Ms. Rita

(JRF) for providing assistance in our endeavor.

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Observed Structures Larval instars

1st 2nd 3rd 4th 5th

Body Types Aphaenogasteroid Pogonomyremocoid Pogonomyremocoid Pogonomyremooid Aphaenogasteroid

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Abdomen 336.4µm 629.0µm 635.0µm 932.6µm 1084.0µm

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